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Triple-negative breast cancers (TNBCs) are heterogeneous and aggressive, with high mortality rates. TNBCs frequently respond to chemotherapy, yet many patients develop chemoresistance. The molecular basis and roles for tumor cell–stromal crosstalk in establishing chemoresistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient–derived xenografts (PDXs) established before and after the development of chemoresistance. Interestingly, the chemoresistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemoresistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibited increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemoresistant model exhibited increased immunosuppression, including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemoresistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established Ras/MAPK-associated gene expression signatures correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T cell–recruiting chemokines (CXCL9/10/11) across patients with TNBC, even in the absence of KRAS mutations. MEK inhibition induced tumor suppression in mice while reversing metabolic and immunosuppressive phenotypes, including chemokine production and gMDSC tumor recruitment in the chemoresistant KRAS-mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemoresistance., Longitudinally-derived TNBC xenografts identify acquired KRAS mutation/MEK activation as a driving feature of CXCL1/2-mediated MDSC recruitment.

Background: Growing evidence supports the critical role of preexisting immune response in triple negative breast cancer (TNBC). However, there are limitations with current evaluation approaches: inability to functionally assess the type of immune infiltration with traditional pathologic evaluation and loss of spatial distribution in conventional high-plex gene or protein expression analyses from the whole tumor section. Here, we report the initial analysis of immune protein expression as a function of spatial distribution and clinical outcomes in TNBC samples. Methods: NanoString GeoMxTM Digital Spatial Profiling (DSP) was used to quantify 39 immune-related proteins in stromal and tumor segments from 44 TNBC samples from the FinXX trial. Samples were matched for patient characteristics, treatment arm (capecitabine vs. 5-fluorouracil), and outcome based on recurrence-free survival (RFS) with 22 samples from patients who recurred and 22 samples from patients with durable RFS. Regions of interest (ROIs) were selected based on expression of cytokeratin (tumor), CD45 (leukocytes), or CD68 (macrophages). Each ROI was segmented into tumor (pancytokeratin-positive area) and stroma (pancytokeratin-negative/nuclear SYTO13-positive area). The general linear model was used for statistical analysis of differential expression with RFS as a categorical variable (recur yes or no). Results: A total of 950 tumor and stroma segments were included in this initial analysis. In both tumor and stroma segments, over-expression of T cell activation markers (CD137, GITR) was associated with better outcome, whereas T cell markers (CD3, CD4, CD8) were not significantly associated with outcome. In tumor segments alone, improved outcome was significantly associated with increased protein expression [> 2-fold change (FC) at p2.0, p2.0, p Citation Format: Saranya Chumsri, Douglas Hinerfeld, Jennifer M. Kachergus, Yaohua Ma, Heather A Brauer, Sarah Warren, Xue Wang, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, E. A. Thompson. Digital spatial mapping of the immune landscape of triple negative breast cancer reveals novel features of immune-tumor cell interaction [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-06.

Background: Growing data established the pivotal role of preexisting immune response in triple negative breast cancer (TNBC). Conventionally, preexisting immune response can be evaluated by quantifying tumor infiltrating lymphocytes mainly in the stroma or gene expression analysis from the whole tumor section. Due to technical challenges with these conventional methods, limited data regarding specific subtypes and spatial distribution of these immune infiltrates are currently available. Methods: NanoString IO360 gene expression analysis and Digital Spatial Profiling (DSP) were used. DSP was used to quantify 29 immune-related proteins in stromal and tumor-enriched segments from 44 TNBC samples from the FinXX trial (NCT00114816) and 335 samples from the Mayo Clinic (MC) cohort of centrally reviewed TNBC (Leon-Ferre BCRT 2018). In FinXX trial, 22 patients with recurrence and 22 patients without recurrence were included. In MC cohort, 217/335 patients received adjuvant chemotherapy while 118 patients had surgery only without adjuvant chemotherapy. Regions were segmented based on pancytokeratin staining. The general linear model was used for statistical analysis of differential expression with recurrence free survival (RFS) as a categorical variable (recur yes or no). Kaplan-Meier (KM) estimates and Cox regression models were also used for analysis. Results: In the FinXX trial, there were 12 out of 29 proteins in tumor epithelial segments (intraepithelial) which were significantly expressed at higher levels among patients who were free of recurrence. These proteins include Beta-2 microglobulin, CD11c, CD20, CD40, CD56, CD8, Granzyme B, HLA-DR, ICOS, PD-L1, PD-L2, and TGFB1. In contrast, merely 5 out of 29 proteins in stromal segments were significantly differentially expressed in these 2 groups of patients. Granzyme B, IDO1, PD-L1, and PD-L2 in stroma were significantly higher and SMA was significantly lower in patients without recurrence. Using Cox regression models, intraepithelial CD56, CD40, and HLA-DR were significantly associated with outcome. When comparing between highest and lowest intraepithelial protein expression by tertile, intraepithelial CD56 (HR 0.12, 95%CI 0.03-0.39, p < 0.001), CD40 (HR 0.13, 95%CI 0.04-0.46, p = 0.002), and HLA-DR (HR 0.24, 95%CI 0.06-0.89, p = 0.032) were significantly associated with improved outcome. However, expression of these same proteins in stroma was not associated with outcome. Using KM estimates, intraepithelial CD56 (p < 0.0001), CD40 (p = 0.0006), and HLA-DR (p = 0.013) were also significantly associated with improved outcome. Nonetheless, RNA expression of these proteins by IO360 from whole tumor sections were not significantly associated with outcome (CD56 p = 0.27, CD40 p = 0.21, HLA-DR p = 0.48). Similar findings with DSP were observed in MC TNBC cohort. Comparing between the highest and lowest quartiles, there were significantly fewer patients who developed recurrence with high protein expression of intraepithelial CD56 (p < 0.001), CD40 (p = 0.002), and HLA-DR (p = 0.006). Conclusions: Using an in-depth analysis with spatially defined context, we identify that there were numerically more intraepithelial immune-related proteins associated with outcome compared to proteins in stroma. Specifically, intraepithelial CD56, CD40, and HLA-DR were significantly associated with improved outcome in both FinXX and MC TNBC cohorts. However, neither expression of these proteins in stroma nor RNA expression from whole tumor were associated with outcome. Our study highlights the impact of spatial biology and the importance of evaluating each potential biomarker in a spatially defined manner. Support: W81XWH-15-1-0292, BCRF 19-161, P50CA116201-9, P50CA015083 Citation Format: Saranya Chumsri, Jodi M. Carter, Yaohua Ma, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, Roberto A. Leon-Ferre, David W. Hillman, Judy C. Boughey, Minetta C. Liu, James N. Ingle, Krishna R. Kalari, Fergus J. Couch, Keith L. Knutson, Matthew P. Goetz, E. A. Thompson. Spatially defined immune-related proteins and outcome in triple negative breast cancer in the FinXX trial and Mayo Clinic cohort [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-02.

Background: The intrinsic immune response in triple negative breast cancer (TNBC) has both prognostic and predictive utility, and immune-related biomarkers are now actionable targets in TNBC. Current modalities for immune profiling are limited by lack of spatial context and precise quantitation. We used quantitative digital spatial profiling (DSP) to 1) characterize the immune architecture of tumoral and stromal regions in TNBC and 2) identify prognostic immune signatures. Methods: From the FinXX trial of adjuvant capecitabine therapy, tumor sections (FFPE) matched for patient characteristics, treatment arm and RFS-based outcome (N=44) were assessed with Nanostring GeoMxTM DSP. Briefly, with 3-plex immunofluorescence (pan-cytokeratin, CD45, CD68 with SYTO-13 dye for nuclei), spatially-defined CD45-enriched or CD68-enriched stromal segments and adjacent tumor segments (N=950) were selected for digital quantitation (NanoString nCounter) of 39 immune proteins (e.g. immune cell profiling proteins: CD3, CD4, CD8, CD11c, CD20, CD56, CD68, HLA-DR, PD-1, PD-L1, Granzyme B), immune drug targets (e.g. VISTA, STING, IDO1), and activation status-related proteins (e.g. ICOS, PD-L2, CD40, CD40L, CD44). Normalized, differentially-expressed (DE) proteins were evaluated, and weighted gene co-expression network analysis (WGCNA) was performed to identify modules of highly co-expressing immune proteins, and association with RFS as a categorical variable was determined. Results: 20 DE proteins in tumor segments were associated with RFS. Among them, those with the highest RFS-associated fold changes (FC) included CD20, CD40, CD56, HLA-DR, Granzyme B, and PD-L2 (FC): 2.1-4.4) (p < 0.05). In contrast, only 4 DE proteins were associated with RFS in stromal segments (Granzyme B, IDO-1, PD-L1, and PD-L2 (FC: 1.5-3.3) (p < 0.05). By WGCNA, 2 tumor-based immune modules [IM1 (lymphocyte infiltration) and IM2 (lymphocyte activation)] were identified based on their co-expression. The top three co-expressed proteins in IM1 were CD4, CD40, and CD45 (r >0.9). The three top co-expressed proteins in IM2 were PD-L1, PD-L2, and Granzyme B (r > 0.85). Together with CD56 expression, IM1 and IM2 comprised a set of 3 intratumoral immune features that associated with RFS. For recurring tumors, all 3 immune features clustered with low scores . Among non-recurrent tumors, two distinct, non-overlapping clusters with inverse characteristics were identified: high IM2, low IM1, low CD56 vs. low IM2, high IM1, and high CD56. Overall, across all 3 immune features, PDL2, CD40 and CD56 expression most strongly associated with RFS. Conclusion: With spatially-defined, precise quantification of immune proteins, we identified 3 immune features, including two distinct immune modules, IM1 and IM2 that correlate with durable RFS. These immune modules of highly co-expressing proteins were associated with RFS only when present in the tumor compartment but not tumor-adjacent, immune cell-rich stroma. Citation Format: Jodi M. Carter, Saranya Chumsri, Yaohua Ma, Xue Wang, Jennifer M. Kachergus, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith A. Perez, Aubrey Thompson. Quantitative digital spatial profiling reveals novel prognostic intratumoral immune signatures in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1004.

510 Background: Several studies have established the critical role of preexisting immune response in triple negative breast cancer (TNBC). Most studies evaluated the tumor infiltrating lymphocytes in stroma. However, limited data are available with regards to the importance of specific subtypes and spatial distribution of these immune infiltrates. Methods: NanoString IO360 gene expression analysis and Digital Spatial Profiling (DSP) were used. DSP was used to quantify 39 immune-related proteins in stromal and tumor-enriched segments from 44 TNBC samples from the FinXX trial (NCT00114816) and 335 samples from the Mayo Clinic (MC) cohort of centrally reviewed TNBC (Leon-Ferre BCRT 2018). In FinXX trial, 22 patients with recurrence and 22 patients without recurrence were included. In MC cohort, 217/335 patients received adjuvant chemotherapy while 118 patients had surgery only without adjuvant chemotherapy. Regions were segmented based on pancytokeratin staining. The general linear model was used for statistical analysis of differential expression with recurrence free survival (RFS) as a categorical variable (recur yes or no). Kaplan-Meier estimates and Cox regression models were also used for analysis. Results: In the FinXX trial, using global gene expression analysis with IO360, there was no signature significantly associated with RFS. However, using DSP, high protein expression of CD56 in the tumor-enriched segments was associated with significant improvement in RFS (HR 0.26, 95%CI 0.09-0.78, p 0.01). Nevertheless, CD56 expression in the stroma (HR 0.66, 95%CI 0.29-1.53, p 0.33) and all segments (HR 0.53, 95%CI 0.23-1.25, p 0.14) was not significantly associated with improved outcome. We further validated these findings in the MC TNBC cohort where intratumoral CD56 expression was associated with a significant improvement in RFS (HR 0.23, p 0.002) but not stromal CD56 (p 0.79). Interestingly, when evaluating the MC TNBC cohort according to receipt of chemotherapy, intratumoral CD56 was associated with improved outcome only in patients who received chemotherapy (p 0.02 vs. 0.07). In both cohorts, higher expressions of intratumoral PD-L1, HLA-DR, and CD8 were associated with improved outcome. Conclusions: Using an in-depth analysis with spatially defined context, we identify that intratumoral CD56-positive NK cells are associated with improved outcome in TNBC. Our study highlights the potential role of NK cells in TNBC and future implications for biomarkers and therapeutic targets.Support: W81XWH-15-1-0292, P50CA116201-9, P50CA015083. Clinical trial information: NCT00114816 .

Background Recent studies have demonstrated a benefit of adjuvant capecitabine, particularly in triple negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy. However, biomarkers to predict which patients are more likely to benefit from capecitabine are needed. Methods The NanoString Breast Cancer 360TM (BC360) and PanCancer ImmunoncologyTM (IO360) panels were used to quantify mRNA expression in TNBC samples in the FinXX trial. FinXX is a phase III trial which randomized high risk patients to receive either 3 cycles of docetaxel followed by 3 cycles of cyclophosphamide, epirubicin, and fluorouracil (Arm A: T+CEF) vs. 3 cycles of docetaxel plus capecitabine followed by 3 cycles of cyclophosphamide, epirubicin, and capecitabine (Arm B: TX+CEX). Gene signature scores were analyzed using prespecified algorithms developed by NanoString. Digital Spatial Profiling was carried out using GeoMX™ platform. A total of 111 TNBC patients were included (57 in Arm A and 54 in Arm B) with 10.2 years median follow up. Results There were 7 cancer- and immune-related gene signatures identified by BC360 and IO360 panels that were significantly associated with improved recurrent free survival favoring an addition of capecitabine. These include cytotoxic cell signature, endothelial signature, mast cell signature, PDL2 gene, immunoproteasome, exhausted CD8 T-cells, and PD1. Spatially-defined analysis of protein expression of a subset of the tumor specimens using the GeoMx platform revealed biological compartment specific (Tumor and/or Stroma) differential expression of immune-related proteins in patients with improved recurrent free survival. Conclusions Analysis of RNA abundance signatures strongly suggests that there are important immune features that are associated with benefit from capecitabine in TNBC. However, analysis of RNA extracted from whole tumor sections lacks spatial discrimination. Using the GeoMx platform for the spatially-defined analysis of protein abundance has provided more insights and has defined specific immune features associated with improved outcome. Clinical trial identification NCT00114816. Legal entity responsible for the study The authors. Funding Finnish Breast Cancer Group. Disclosure D.A. Hinerfeld: Full / Part-time employment: Nanostring. H.A. Brauer: Full / Part-time employment: Nanostring. T. Nielsen: Licensing / Royalties: Nanostring. All other authors have declared no conflicts of interest.

Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes between tumor and stromal cells involved in developing chemo-resistance are under-explored. Here we report studies of paired TNBC patient-derived xenografts (PDX) established before and after chemo-resistance. Despite significant genetic similarities, the chemo-resistant PDX model harbored a rare constitutively-active KRASQ61R mutation which was not present in the chemo-naive PDX. Further analysis demonstrated that the chemo-resistant KRAS-mutant model exhibited altered gene expression changes including increased expression of CXCR2-ligands CXCL1 and CXCL2, which are responsible for recruiting immune cells to tumors. These expression patterns were largely inhibited in vivo by MEK inhibitor (MEKi) treatment. Moreover, in breast cancer cell lines, CXCL1, CXCL2, and granulocyte macrophage-colony stimulating factor (CSF2, stimulates granulocyte and macrophage differentiation from hematopoietic precursor cells, including immunosuppressive myeloid cells) transcripts were also downregulated by MEKi. Notably, chemo-resistant KRAS-mutant tumors harbored increased Gr1+ and Arginase-1+ cells, consistent with recruitment of immunosuppressive M2-like macrophages and/or myeloid-derived suppressor cells (MDSCs), which was inhibited by MEKi. Further experiments demonstrate that CD45+CD11b+Ly6G+ MDSC accumulation in tumors can be inhibited by MEKi treatment alone, or by CXCR2 inhibition, suggesting that the effects of MEK inhibition on MDSC recruitment are CXCL1/2-dependent. Confirming the translational relevance of these findings, in >200 murine and >1000 human breast tumors, Ras/MAPK transcriptional activity correlated with myeloid-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11), even in the absence of activating KRAS mutations. The association with Ras/MAPK activity was also confirmed using immunofluorescence to quantify MHC-II-low myeloid cells in 80 post-chemotherapy TNBC tumors. Importantly, MEKi and chemotherapy combination treatment reversed immunosuppressive cell accumulation and metabolic phenotypes exemplified by altered optical redox ratios (NAD(P)H/FAD) in the chemo-resistant KRAS mutant tumors, resulting in tumor growth suppression in mice. MEKi treatment also reduced redox ratios in 3D cultures of breast cancer cell lines further suggesting that MEK inhibition targets multiple oncogenic processes in breast cancer. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistant disease. Citation Format: Derek A. Franklin, Joe T. Sharick, Paula I. Ericsson-Gonzalez, Violeta Sanchez, Phillip T. Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko. MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1511.

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) activate innate immunity to increase tumor immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including inefficient transport to the cytosol where STING is localized. We recently developed STING-activating nanoparticles (STING-NPs), polymer vesicles designed for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2’3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumorally-administered (IT) STING-NPs significantly enhance the therapeutic efficacy of cGAMP and improve response to immune checkpoint inhibition (ICI) in established murine B16 melanoma tumors. However, the immunologic effects of STING-NPs have not been well characterized in tumors or lymphatic tissue. We utilized Digital Spatial Profiling (DSP) to identify immunologic responses in the injected tumor, a distal tumor, and both tumor draining lymph nodes (TDLN) 48 hours after a single IT injection of STING-NP or three injections of STING-NP co-administered with systemic ICI (anti-PD-1/CTLA-4). DSP analysis permits simultaneous detection of over 30 protein markers in distinct spatial regions of interest (ROI; n=2-4 per tumor/TDLN) or cell types in tissue sections. After a single STING-NP injection of B16 tumors, B7-H3, an immunosuppressive T cell checkpoint, was significantly induced in tumors compared to PBS or free cGAMP. More strikingly, in T cell-rich regions of the TDLN, STING-NP dramatically increased B7-H3, GZMB, and S100A9 while suppressing VISTA and CD73 expression. After multiple injections of STING-NP, B7-H3, S100A9, CD73, Foxp3, and beta-catenin were substantially upregulated in the injected tumor, with more modest fold changes in distal tumors. Co-treatment with systemic ICI in part abrogated this effect. In contrast, STING-NP-induced changes in GZMB, CD4, and CD8a expression were observed regardless of ICI treatment, and were generally equivalent in both treated and distal tumors, suggesting an abscopal effect. Both the treated and distal TDLN showed striking S100A9 increases which was preferential, but not exclusive, to T cell-rich vs. B cell regions. This effect was exacerbated with the combination of STING-NP and ICI therapy. B7-H3 upregulation in response to STING-NP (regardless of ICI) was exclusive to treated TLDNs vs. distal TDLNs. B7-H3 was expressed in both B and T cell regions, but was nearly two-fold higher in B cell-rich regions. Upregulation of GZMB (5-10 fold over free cGAMP alone) was observed in both treated and distal TDLNs. Markers of T cell activation were observed after STING-NP treatment, including moderate abscopal effects in synchronous distal tumors. In TDLNs, STING-NP robustly induced S100A9 and B7-H3, which may be immunosuppressive escape mechanisms that could be targeted to enhance responses. Citation Format: John T. Wilson, Daniel Shae, Paula I. Gonzalez-Ericsson, Violeta Sanchez, JingJing Gong, Yan Liang, Douglas Hinerfeld, Joseph M. Beechem, Justin M. Balko. Digital spatial profiling of molecular responses to nanoparticle STING agonists identify S100A9 and B7-H3 as possible escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4978.

3142 Background: Recent studies have demonstrated a benefit of adjuvant capecitabine, particularly in triple negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy. However, biomarkers to predict which patients are more likely to benefit from capecitabine are needed. Methods: The nanoString Breast Cancer 360 (BC360) and PanCancer Immunoncology (IO360) panels were used to quantify mRNA expression in TNBC samples in the FinXX trial. FinXX is a phase III trial which randomized high risk patients to receive either 3 cycles of docetaxel followed by 3 cycles of cyclophosphamide, epirubicin, and fluorouracil (Arm A: T+CEF) vs. 3 cycles of docetaxel plus capecitabine followed by 3 cycles of cyclophosphamide, epirubicin, and capecitabine (Arm B: TX+CEX). Gene signature scores were analyzed using prespecified algorithms developed by nanoString. Digital Spatial Profiling was carried out using GeoMX platform. Results: A total of 111 TNBC patients in FinXX trial were included (57 in Arm A and 54 in Arm B) with 10.2 years median follow up. There were 7 cancer- and immune-related gene signatures identified by BC360 and IO360 panels that were significantly associated with improved recurrent free survival favoring an addition of capecitabine. These include cytotoxic cell signature (HR 0.37, 95%CI 0.15-0.92, p 0.03), endothelial signature (HR 0.18, 95%CI 0.04-0.83, p 0.03), mast cell signature (HR 0.43, 95%CI 0.21-0.88, p 0.02), PDL2 gene (HR 0.29, 95%CI 0.09-0.99, p 0.05), immunoproteasome (HR 0.34, 95%CI 0.13-0.89, p 0.02), exhausted CD8 (HR 0.29, 95%CI 0.09-0.97, p 0.04), and PD1 (HR 0.44, 95%CI 0.20-1.02, p 0.05). Conclusions: Analysis of RNA abundance signatures strongly suggests that there are important immune features that are associated with benefit from capecitabine in TNBC. However, analysis of RNA extracted from whole tumor sections lacks spatial discrimination. We anticipate that a more detailed, spatially-defined analysis of protein abundance, using the novel NanoString GeoMX platform, will provide more insights and define specific immune features associated with improved outcome. Additional results of GeoMX will be reported at the meeting. Clinical trial information: NCT00114816.

e14218 Background: Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues enables a better understanding of biological systems in many research areas, including immunology and oncology. However, it has proven difficult to perform such studies in a highly multiplexed manner. To address this unmet need, we have developed a novel optical-barcode based microscope and tissue-sampling platform designed to simultaneously analyze hundreds of proteins or mRNAs on a single FFPE section from distinct tissue spatial regions. Methods: Using colorectal cancer FFPE sections we spatially resolve protein and mRNA expression of immune-oncology targets and present a series of modalities and associated applications for the GeoMx Digital Spatial Profiler platform. Results: Microsatellite stable (MSS) or instable (MSI) colorectal tumors will be characterized to evaluate active and suppressive immune mechanisms in both immune dense regions and tumor versus stroma by utilizing segment profiling. Regions from the invasive margins and tumor centers will be investigated with contour profiling to define different immunosuppressive and activated immune phenotypes. Evaluation of tumor versus stroma will also identify pathways related to each compartment that are different between MSI and MSS tumors. These techniques can be used to discover drug mechanism of action or immune activation status, as well as to facilitate prediction of treatment response and disease progression or investigation of specific rare cell populations’ molecular profiles. Conclusions: This study will demonstrate multiplexed pathway-level protein and gene expression analysis from discrete regions within a colorectal tumor (tumor center and immune invasive margin), enabling systematic interrogation of immune activity in FFPE samples. Further studies could eventually lead to the identification of unique localized immune characteristics to guide combination therapeutic approaches.

e15139Background: Spatial characterization of immune microenvironment within tumors enables a better understanding of immunology and oncology. However, it has proven difficult to perform such studi...

Because cerebrospinal fl(CSF) abnormalities in presymptomatic subjects with PSEN1 (presenilin 1) mutations may be observed 4 to 12 years prior to the estimated age at onset, it is possible to test putative therapies on the CSF analytes that correlate with neurodegeneration during this presymptomatic window of clinical opportunity. It is also possible to test the same therapy on a comparison group with increased risk status conferred by both hyperlipidemia and heterozygosity for apolipoprotein Ee4. To our knowledge, the only putative therapy thus far tested in such a common design has been statin therapy. The results of these tests show increases in soluble amyloid precursor protein (sAPP)α correlating with statininduced decreases in serum cholesterol levels in the non-PSEN1 subjects. This result could be one functional correlate for part of the substantial risk reduction for late onset Alzheimer’s disease recently reported in the Rotterdam study, a large, long-term prospective statin trial. Statin therapy signifi cantly decreased both sAPPα and sAPPβ in presymptomatic PSEN1 subjects. Initially, elevated phospho-tau levels in PSEN1 subjects did not further increase during the 2 to 3 years of statin therapy, possibly indicative of a prophylactic eff ect. These results suggest that large and longer term trials of statin therapy correlating changes in CSF biomarker levels with clinical course may be warranted in both presymptomatic PSEN1 and non-PSEN1 subjects.

KDR, also known as VEGFR2, is a target of several VEGFR inhibitors, however, only bevacizumab, which targets the VEGF ligand, is FDA approved for lung cancer. Since an activating mutation in KDR was identified in 70% of lung adenocarcinoma patients we sought to identify the number of available clinical trials targeting KDR in lung adenocarcinomas and/or solid tumors, thereby increasing therapeutic options and treatment accessibility for these patients. DNA from thirteen lung cancer FFPE samples was sequenced on Illumina HiSeq 2500 or MiSeq sequencers using a CLIA-certified 358-gene targeted sequencing assay. FASTQ files generated from Illumina's CASAVA software were submitted to a Clinical Genome Analytics (CGA) data analysis pipeline to perform automated read quality assessment, alignment, and variant calling. Clinical reports were written for each sample through extensive literature and clinicaltrials.gov searches. Thirteen lung biopsies were obtained and subdivided based on pathology reports, into 10 lung adenocarcinomas, 1 mixed NSCLC/SCLC, 1 small cell carcinoma, and 1 squamous carcinoma. KDR Q472H was identified in 7/10 lung adenocarcinomas and 1/1 mixed NSCLC/SCLC, but not in the small or squamous cell carcinomas. KDR encodes Vegfr-2, a receptor tyrosine kinase overexpressed in a variety of solid tumors. KDR Q472H is an activating missense mutation, which increases phosphorylation and activation of Vegfr-2, thereby promoting increased microvessel density in lung tumor tissues. At the time of clinical report writing, there were a total of 47 actively recruiting clinical trials targeting KDR and 4 trials requiring the patient to have a molecular alteration of KDR. Further breakdown of these trials identified the use of 33 off-label VEGFR inhibitors, 5 investigational pan-VEGFR inhibitors, and 5 investigational specific KDR (VEGFR2) inhibitors. Next-generation sequencing of 358 genes from lung cancer biopsies identified a common KDR activating mutation, Q472H, in 70% of lung adenocarcinomas. Searching clincialtrials.gov by pan-VEGFR and specific KDR inhibitors identified 47 actively recruiting trials, thus increasing the accessible repertoire of treatment options for lung adenocarcinoma patients. Citation Format: Susan M. Mockus, Christopher S. Potter, Grace A. Stafford, Guruprasad Ananda, Douglas Hinerfeld, Gregory J. Tsongalis. Targeting KDR mutations in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2015-73

The Jackson Laboratory Cancer Treatment Profile™ (JAX-CTP™) is a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable gene variants in solid tumors to inform the selection of targeted therapeutics for cancer treatment. We will describe the design of the 358- gene panel, analytical validation, and the curation and clinical reporting of actionable variants. Selection of the gene panel was based on known drug targets, casual implications in cancer, and a thorough pathway analysis. DNA is extracted from FFPE tumor samples and using hybrid capture, the genes of interest are enriched and sequenced on Illumina HiSeq 2500 or MiSeq sequencers. FASTQ files generated from Illumina's CASAVA software are submitted to the JAX Clinical Genome Analytics (CGA) data analysis pipeline to perform automated read quality assessment, alignment, and variant calling. Identified variants are then submitted for clinical curation using a combination of the in-house JAX Clinical Knowledgebase (CKB) and the external Genetic Variant Annotation (GVA) from CollabRx. Once clinically annotated, the variants are graded relative to their clinical utility for the specific tumor type and compiled into a clinical report to inform patient treatment. Extensive analytical validation, following CAP guidelines, was conducted to assess limit of detection, accuracy, precision, sensitivity, and specificity of the assay. The summarized optimized sensitivity of the assay is a minimum coverage of samples at 300X, a limit of detection of 10% for SNP’s/indels and ≥6 copies for CNV’s, and an average of 3-4 actionable variants per sample. The challenges of interpreting gene variants for clinical actionability and for establishing an analytically valid bioinformatic pipeline will be discussed in-depth. Citation Format: Susan M. Mockus, Guruprasad Ananda, Micaela Lundquist, Vanessa Spotlow, Al Simons, Talia Mitchell, Grace A. Stafford, Christopher S. Potter, Vivek Philip, Timothy Stearns, Anuj Srivastava, Mary Barter, Lucy Rowe, Joan Malcolm, Carol Bult, Radha Krishna Murthy Katuturi, Karen Rasmussen, Douglas Hinerfeld. Design, validation, and interpretation of an NGS assay for actionable variants in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2015-4816

e22117 Background: Matching the mutational profile of a patient’s tumor with appropriate targeted agents is a goal of personalized medicine in oncology. While the targeted therapeutic armamentarium...